In eukaryotes, membrane contact sites (MCSs), at which the endoplasmic reticulum (ER) and another organelle come into close proximity, play key roles in lipid homeostasis, but the processes that occur at these sites remain poorly understood at the molecular level. Our approach to better understanding the function of contact sites and the regulation of activities that take place there is to characterize the protein machinery residen at such sites, focusing on contacts between the ER and the plasma membrane (PM). One process recently identified as important for lipid homeostasis as mediated by MCSs is the non- vesicular transport of lipids by lipid transfer proteins. We will combine structural, biochemical, and cell- based studies to characterize lipid transporters at ER-PM contact sites, addressing which proteins participate in lipid transfer, how they are targeted to these sites, which lipids thy transport, how they recognize their lipid ligands, and how transport by these proteins might be regulated. Aim 1 focuses on the protein TMEM24, present only in metazoans and recently identified as a regulator of insulin secretion. Preliminary data indicate TMEM24 as an ER-PM contact resident with a lipid-binding module that specifically associates with phosphatidylinositol (PI). In probing the mechanisms that underlie TMEM24 activity at contact sites, we will test the hypothesis that TMEM24 transports PI from its place of synthesis in the ER to the PM, where PI can be rapidly converted to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. PI(4,5)P2 is a major PM signaling lipid critical for vesicle docking and priming, and we propose that the role of TMEM24 in secretion is to supply PM PI(4,5)P2. In Aim 2, we will apply a similar approach to characterize proteins in the lipid transfer proteins anchored at membrane contact sites (LAM) family, newly identified in yeast as key for the transport of sterol, a major PM component. We will expand investigations of the LAM family to the metazoan proteins, identifying which members of this family are targeted to ER- PM contacts, probing whether they play the same critical role as in fungi, and structurally and functionally dissecting the molecular mechanisms by which they are targeted to ER-PM contacts and transport cholesterol to the PM.